Primary productivity

Determination of the photosynthetic parameters of phytoplankton. At the DAGON-Flux stations, measurements were made between 0 and 500m using a PhytoPAM. Fluorescence measurements (Fv/F0) and fast kinetics of light gradient responses are used to calculate Pbm, alpha value and light photo-adaptation index (Ik).

Primary productivity

Collaboration with M. Acker and D. Repeta (WHOI) Seawater samples were collected (0-250m) at the DAGON-Flux stations to fill 'alive' and 'killed' triplicats. Samples spiked with 33PO4 were placed in deck incubators for 3h (5m) or 4h (50m). Then, samples were filtered onto 2 different 25 mm 0.2 µm isopore filters, which were counted back in the lab to deduce phosphate uptake rate and reduction rate after extraction, separation, and fractionation of the different reduced phosphorus compounds.

Primary productivity

Measurements at DAGON-Flux stations of incorporations of 15N (NO3, NH4, N2) and 13C at 6 depths (5, 10, 20, 20, 50, 75, 100 m). Incubations for 24 hours in simulated in situ on the ship foredeck. Samples were filtered on precombusted GFF filters and stored dry until analysis. Some filtrates were conserved (ammonium-enriched samples).

Primary productivity

Collaboration with S. Psarra (HCMR) Measurements at DAGON-Flux stations of 14C incorporation by size fractionation at 6 depths (5, 10, 20, 50, 75, 100 m). Incubations for 24 hours by in situ simulated conditions on the deck incubators on the ship foredeck. After filtration, acidification and drying, a scintillation cocktail is added before passing in a scintillation counter. The rates are deducted taking into account the T0, and the carbon fixation in the dark.

Phytoplankton pigments (eg chloroph

Water sampled at TAHRUN-Particulate and Phytofloat stations with Niskin bottles at chosen depths according to the water vertical profile properties (up to 12 depths per profile). Filtrations on Whatman GF/F 25mm diameter filters. Immediate conservation in liquid nitrogen and storage at -80ºC. Analysis proceeded on land by HPLC by the national service SAPIGH (http://www.sapigh.obs-vlfr.fr/)

Seston

The Underwater Vision Profiler (UVP) allows to quantify the vertical distribution of macroscopic particles and zooplankton > 100 µm in size. The smaller size limit is fixed by optical resolution, whereas the larger size limit is determined by the volume of water (1.02 L) illuminated per image. The sampling of the UVP is 6 Hz and was mounted on the rosette. Processing on ImageJ to get profiles of particles abundance per size classes, and vignettes of particles and zooplankton organisms.

Seston

S. Chaikalis (HCMR) Collaboration. The LISST-Deep instrument obtains in-situ measurements of particle size distribution, optical transmission, and optical volume scattering function. The treatment requires the definition of a background spectrum that is obtained at the deep stations at the clear water minimum level. The size spectrum and total volume concentration are obtained by inverting the diffraction spectrum.

Dissolved organic matter (inc DOC)

Collaboration with A. Gogou (HCMR). Lipid Biomarkers and stable isotopes (delta13C et delta15N). Water sampled from Niskin bottles at selected depths according to the vertical profile properties (up to 6 depths per profile). Sampled volume = 48 - 96 L for the determination of n-alkanes, n-alkanols, sterols, alkenones, diols and keto-ols, fatty acids and for stable isotopic composition of C and N (delta13C and delta15N). Immediate filter storage at -20ºC.

Dissolved organic matter (inc DOC)

Seawater samples collected at ZEUS-Dissolved stations on 19 levels between surface and bottom for Dissolved Organic Matter analysis (DOC, DON and DOP). DOC analyzed by high temperature catalytic combustion (Shimatzu TOC-L) and DON and DOP by wet oxidation then measured by Technicon autoanalyzer

Pelagic bacteria/micro-organisms

Water samples are taken directly from Niskin bottles at ZEUS-Dissolved stations, in 15 mL Falcon tube and then subsampled in 1 cryotube of 2 mL (send to Banyuls sur Mer for heterotrophic prokaryote determination) and 1 cryotube of 4 mL (send to Marseille for pico- nano- plankton determination). They are fixed with glutaraldéhyde, flashfrozen after 15 min. and stored at -80°C until analysis.

Pelagic bacteria/micro-organisms

Collaboration with Psarra S. (HCMR) MIXO-grazing by flagellates (heterotrophic and mixotrophic) Short-term grazing experiments were conducted using fluorescence-labelled bacteria (FLB) as prey at DAGON-Flux stations. Triplicats are filled for each depth and FLBs added to this in ca 15% of the natural abundance. The bottles are placed (40 min) in thermoregulated incubators on the deck simulating in situ light conditions.

Pelagic bacteria/micro-organisms

Heterotrophic prokaryotic production incorporation of tritiated leucine into proteins. Inoculation with a mix of 3H-leucine and cold leucine, final concentration 20 nM. Note that all incubations are made in the dark, at in situ temperature. Incubation time lasted 3 to 6 hours according the expected activities. Samples are treated with the centrifuge technique, 3 runs of centrifugations including a 5% TCA rinse and an 80 % ethanol rinse.

Pelagic bacteria/micro-organisms

Ectoenzymatic activity (alkaline phosphatase, phosphodiesterase) Addition of fluorogenic substrates Methylumbellyferyl phosphate disodium salt (MUF-P), bis Methylumbellyferyl phosphate sodium salt (bis MUF P) in total sea water, incubations in the dark at in situ temperature, lecture of rates by spectrofluorimetry. Phosphatase and diesterase concentration kinetic (Km and Vm) derived from non linear regression fit of the Michaellis Menten equation.

Pelagic bacteria/micro-organisms

Samples collected from Niskin bottles (TAHRUN-Particulate stations) were successively filtered on 3 µm polycarbonate membrane (attached microbial communities) and 0,2 µm sterivex system (free microbial communities) and then stored at -80°C for future analysis. The extracted DNA (DNeasy Blood & Tissue Kits, Qiagen) samples were quantified using a fluorimetric method (Invitrogen? Quant-iT? PicoGreen? dsDNA Assay Kit) and the hypervariable regions of the 16S rRNA gene sequenced (illumina)

Phytoplankton

HCMR collaboration Nalgene bottles are directly filled from the Niskin bottles at DAGON-Flux stations for the determination of phytoplankton species and cell abundance. The samples are fixed with glutaraldehyde (final concentration 0,2%) The identification of phytoplankton species and the enumeration of phytoplankton cells (> 5?m) are performed in 100 mL sedimentation chambers with an inverted light microscope (OLYMPUS IX70), according to the standard sedimentation method.

Zooplankton

At the TAHRUN-Particulate stations, series of nets (Triple WP2 nets: 2 nets of 200 µm, 1 net of 100 µm and Multinet Hydrobios: 5 nets of 200 µm) were carried out in order to have access to biomasses (CHN), size structures (Zooscan and Flowcam), abundances (stereoscope), diversities, respiratory frequencies, biochemical compositions, energy contents, trophic levels and genetic contents by metabarcoding

Particulate organic matter (inc POC

Collaboration with K. Parinos (HCMR) TEP, Coomassie Blue stained particles, Carbohydrates, Proteins Water sampled from Niskin bottles at selected 10 depths per profile at TAHRUN-Particulate stations was taken for TEP, Coomassie Blue stained particles and carbohydrate and protein analysis. Filtration of the sampled volume on Polycarbonate 25mm diameter filters with 0.4 µm porosity. Immediate storage at -20ºC.

Particulate organic matter (inc POC

Samples from TAHRUN-Particulate stations on 12 levels and filtration on 25 mm diameter GFF filters previously weighed. The filters, rinsed with milliQ water, are dried in the oven and then stored in PetriSlides before being analyzed on a CHN mass spectrometer back in the laboratory.

Biochemical meas. (eg,lipids,amino

Sampled from TAHRUN-Particulate++ stations are filtered through 0.2 µm under laminar flow hood. Phosphate are analysed on board by CFA-LWCC technique (auto-analyzer system coupled to a 1 m length LWCC and connected to a USB-4000 spectrophotometer). AP-DOP measurements: Prior to analysis, subsamples are spiked with a purified alkaline phosphatase and incubated at 30°C during 4h. AP-DOP concentrations are calculated from the difference in phosphate before and after incubation

Biochemical meas. (eg,lipids,amino

Collaboration with M. Acker and D. Repeta (WHOI) CP lyase activity At the DAGON-Flux stations, triplicats of 125 mL seawater were spiked with a specific fluorescent reactant and a phosphonate is chemically added to a fluorescent probe, and incubated (24h). Samples were filtered onto a ENV+ SPE column, which will be eluted with methanol back in the lab. The extract are injected onto a C18 column for separation and deduction of the relative activity of the CP lyase enzyme.

Current profiler (eg ADCP)

Shipboard ADCP

Current profiler (eg ADCP)

The system consisted of an upward-looking (slave) and a downward-looking (master) ADCP providing each a current profile over a range of 100 m maximum. Both ADCP are mounted on the rosette. The sampling rate is 1 Hz. It measures on cells of 10 m and with 1 ping per set with a maximum range of 100 m. Data are processed with the GEOMAR LADCP software (Version 10.14) uses ship ADCP (S-ADCP), navigation, and CTD profiles acquired simultaneously to reconstruct current profiles.

Current profiler (eg ADCP)

Hull monted ADCPs . Continuous recording along ship track. The OS 38 measures on cells of 24 m and with 1 ping per set with a maximum range of 1000 m. The OS 150 measures on cells of 8 m and with 1 ping per set with a maximum range of 400 m. Data processing is carried out using the LPO CASCADE software (Version 7.2)

Other physical oceanographic meas.

Gravity measurements

Single-beam echosounding

Single channel seismic reflection

Water bottle stations

At stations ZEUS-Dissolved, TAHRUN-Particulate, et DAGON-Flux, a rosette of 24 bottles was used during the cruise. Only 21 bottles were mounted to allow the UVP to be connected. The profiles were carried out from surface to bottom on the 97 prospected stations (126 CTD casts). Note = ZEUS stations include TAHRUN and TAHRUN stations include DAGON

CTD stations

At stations HYDRO, ZEUS-Dissolved, TAHRUN-Particulate, and DAGON-Flux, CTD fixed on the rosette deployed with the vessel's starboard winch. The equipments are: CTD Seabird SBE 9 PLUS Deck Unit Seabird Oxygen probe Seabird SBE 43 Temperature sensor x2 Seabird SBE 3 Conductivity sensor x2 Seabird SBE 4 Pump x2 Seabird SBE 5 Pressure sensor Seabird Transmissiometer C-Star WetLabs Turbidimeter Seapoint TUR002 Fluorimeter Chelsea Aquatracka III

Optics (eg underwater light levels)

Collaboration with Nektarios Spyridakis from HCMR. The Submersible Radiance sensor was installed on the Submersible Optics Platform for each cast or installed on the Starboard side of the vessel to collect Ocean Colour surface reflectance measurements. The LISST-Deep instrument obtains in-situ measurements of particle size distribution, optical transmission, and the optical volume scattering function. The LISST-Holo2 stores holograms for study of flocs, plankton, and other particles in water.

Oxygen

Oxygen was collected once per day in association with AT-CT and pH, to adjust the SBE43 profiles (surface to bottom) and measured on board with photometric system using the Winkler titration method

Phosphate

Samples (x3) for nutrient analysis are taken at ZEUS-Dissolved stations directly from the rosette in 20 mL scintillation vials and analyzed immediately on board on a Technicon autoanalyzer (duplicate) for the various nutrients including PO4 (and NO2+NO3, NO2, Si(OH)4). The last 20 mL vial is quickly frozen at -20°C for analysis after return to the laboratory.

Nitrate

Samples (x3) for nutrient analysis are taken at ZEUS-Dissolved stations directly from the rosette in 20 mL scintillation vials and analyzed immediately on board on a Technicon autoanalyzer (duplicate) for the various nutriens including NO3 (and NO2+NO3, PO4, Si(OH)4). The last 20 mL vial is quickly frozen at -20°C for analysis after return to the laboratory.

Nitrite

Samples (x3) for nutrient analysis are taken at ZEUS-Dissolved stations directly from the rosette in 20 mL scintillation vials and analyzed immediately on board on a Technicon autoanalyzer (duplicate) for the various nutrients including NO2 (and NO2+NO3, PO4, Si(OH)4). The last 20 mL vial is quickly frozen at -20°C for analysis after return to the laboratory.

Silicate

Samples (x3) for nutrient analysis are taken at ZEUS-Dissolved stations directly from the rosette in 20 mL scintillation vials and analyzed immediately on board on a Technicon autoanalyzer (duplicate) for the various nutrients including Si(OH)4 (and NO2, NO2+NO3, PO4). The last 20 mL vial is quickly frozen at -20°C for analysis after return to the laboratory.

Alkalinity

AT-CT was collected once per day in association with oxygen and pH samples (surface to bottom) and stored in the cold room before sending to SNAPO-CO2 (Paris) analytical service. CT and AT are measured via potentiometric titration (using a closed cell).

pH

pH samples were collected once per day in association with oxygen and AT-CT (surface to bottom) and measured on board with a spectrophotometric instrument (SOMLIT protocol).

Other dissolved gases

Collaboration with M. Acker and D. Repeta (WHOI) Methane concentration At the DAGON-Flux stations, seawater was collected (11 depths) directly into 70 mL combusted glass bottles flushed with 2 times the bottle volume and killed with 200 µL saturated HgCl2 then sealed and stored in the dark. Back in the lab, methane concentrations were measured by gas chromatography.

Other dissolved gases

Collaboration with M. Acker and D. Repeta (WHOI) Methane isotopes At 3 DAGON-Flux stations, seawater was collected (8 depths) directly into 500 mL combusted glass bottles flushed with 2 times the bottle volume and killed with 250 µL saturated HgCl2 then sealed and stored in the dark. Back in the lab, methane concentrations were measured by a gas chromatograph combustion isotope ratio mass spectrometer system.

Surface measurements underway (T,S)

The thermosalinometer circuit of the RV Pourquoi Pas? was activated as soon as the departure from La Seyne sur Mer and operated throughout the cruise in greek and French waters.

Carbon dioxide

AT-CT was collected once per day in association with oxygen and pH samples (surface to bottom) and stored in the cold room before sending to SNAPO-CO2 (Paris) analytical service. CT and AT are measured via potentiometric titration (using a closed cell).

Ammonia

Ammonia (NH4 ± 2 nM) is measured between the surface and 500 m (plus 1 point close to the bottom) at 12 depths at ZEUS-Dissolved stations. Samples are taken in ultra-clean scintillation vials and NH4 concentrations are measured on board by fluorimetry using a Denovix spectrofluorimeter.

Incident radiation

Collaboration with N. Spyridakis (HCMR). The Trios sensor was installed on the submersible optical platform for each profile or on the starboard side of the vessel to collect reflectance measurements of the ocean surface.

Routine standard measurements